RESTRICTIVE EPITOPE PEPTIDE OF MAJOR HISTOCOMPATIBILITY COMPLEX B2 OF H9N2 SUBTYPE AVIAN INFLUENZA VIRUS AND APPLICATION THEREOF

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to Chinese Patent Application No. 202210449829.4, filed on Apr. 27, 2022, the contents of which are hereby incorporated by reference.

INCORPORATION BY REFERENCE STATEMENT

This statement, made under Rules 77(b)(5)(ii) and any other applicable rule incorporates into the present specification of an XML file for a “Sequence Listing XML” (see Rule 831(a)), submitted via the USPTO patent electronic filing system or on one or more read-only optical discs (see Rule 1.52(e)(8)), identifying the names of each file, the date of creation of each file, and the size of each file in bytes as follows:

• • File name: 347_004_2023_3574 sequence listing
  • Creation date: Apr. 20, 2023
  • Byte size: 427,819 bytes

TECHNICAL FIELD

The present application relates to the technical field of genetic engineering, and in particular to a restrictive epitope peptide of a major histocompatibility complex (MHC) B2 of an H9N2 subtype avian influenza virus (AIV) and an application thereof.

BACKGROUND

Avian influenza virus (AIV) is a kind of segmented ribonucleic acid (RNA) virus belonging to the genus Influenza virus A of the family Orthomyxoviriade, with hosts ranged from various avian species to mammals including humans. Based on the serological differences in Hemagglutinin (HA) and Neuraminidase (NA), AIV can be classified into 18 HA subtypes and 11 NA subtypes, with H9N2 subtype AIV being prevalent in poultry in China. Despite of being a low pathogenic avian influenza, H9N2 subtype AIV can still cause significant economic losses by reducing egg production or co-infecting chickens with other pathogens in poultry, making it essential to strengthen prevention, control and research on H9N2 subtype AIV.

Currently, inactivated vaccines are mainly used for preventing and controlling this virus; however, the virus is likely to mutate and evade from the recognition of antibodies as a result of prolonged immune selection pressure, which leads to insufficient protection across subtypes by the specific antibodies produced by the vaccine. Therefore, the development of vaccines with broader coverage and longer-lasting protection is important for preventing and controlling avian influenza.

Extensive studies have shown that influenza-specific CD8⁺ T cells not only participate in viral clearance but also provide cross-protection against other subtypes of influenza viruses, as demonstrated by Dai et al. who found that CD8⁺ T cell response plays an important role in fighting AIV infection by comparing key protective factors generated by H9N2 AIV infection and vaccine immunization-induced immune response in pathogen-free chickens; while Seo et al. found that chickens infected with H9N2 AIV showed higher survival rates in H5N1 AIV infection assays and the survival rate of chickens infected with H5N1 AIV was increased by subsequent injection with activated H9N2 AIV-specific CD8⁺ T cells. In summary, the development of a vaccine that can induce T-cell immune responses is of great importance for the prevention and control of H9N2 AIV.

Immunogenic epitopes are prerequisites for inducting immune effects in T cells. By March 2022, the Immune Epitope Database (IEDB) showed a total of 34 T-cell epitopes of AIV against chickens, of which 24 T-cell epitopes had been functionally validated for immunogenicity, including 22 CD8⁺ T-cell epitopes and 2 CD4⁺ T-cell epitopes on nucleoproteins, polymerase proteins, matrix protein 1 and haemagglutinin, covering three subtypes of H5N1, H5N8 and H7N1, but no epitopes have been reported for the H9N2 subtype of AIV. Therefore, a systematic screening for immunogenic AIV epitopes is required to address the current long-term prevalence of H9N2 subtype AIV.

Antigenic epitopes are recognized by T cell receptors (TCRs) through binding to major histocompatibility complex (MHC) class I molecules; however, MHC class I molecules are polymorphic and different MHC class I molecules can bind different antigenic epitopes even for the same pathogen, so it is important to clarify the restriction of the MHC while screening for antigenic epitopes. Currently, chickens can be classified into 29 haplotypes from B1 to B29 based on the gene sequence of the MHC B gene region, of which haplotype B2, a common haplotype, has been widely reported to show resistance to certain diseases and is an excellent material for experimental studies on AIV and vaccine development.

SUMMARY

The objectives of the present application are to provide a restrictive epitope peptide of a major histocompatibility complex (MHC) B2 of an H9N2 subtype avian influenza virus (AIV) and an application thereof, so as to solve the problems existing in the prior art. According to the present application, an animal model of H9N2 subtype AIV infection in B2 haplotype chickens is established to demonstrate the important role of cellular immune response in the resistance of B2 haplotype chickens to AIV infection, and potential epitopes in H9N2 subtype AIV viral proteins are systematically screened using the binding motif of B2 haplotype MHC class I molecular, and finally peptide epitopes with immunogenicity are identified through functional assays to facilitate the development of AIV epitope vaccines.

To achieve the above objectives, the present application provides technical schemes as follows:

• • the present application provides a restrictive epitope peptide of an MHC B2 of an H9N2 subtype AIV, where the restrictive epitope peptide has an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 or SEQ ID No.4.

The present application also provides an application of the restrictive epitope peptide in preparing vaccine against H9N2 subtype AIV.

The present application also provides a vaccine against H9N2 subtype AIV, including the restrictive epitope peptide.

The present application achieves the following technical effects:

• • according to the present application, firstly, H9N2 subtype AIV (A/Chicken/Hunan/HN/2015) strain is used to infect B2 haplotype (BW/G3) SPF chickens, and the success of the infection model is determined by examining cloacal virus shedding, oropharyngeal virus load, changes in T-cell subtypes in peripheral blood mononuclear cells (PBMC) and expression of immune-related genes in PBMC to act as a follow-up test material; then, candidate peptide epitopes that are potentially immunogenic against H9N2 subtype AIV are screened based on the motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-X-V/I/L) of B2 haplotype chicken MHC class I molecule identified in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, and the immunogenicity of the above peptides is finally verified by the ELISpot assay to identify valid T cell epitope.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to explain the embodiments of the present application or the technical schemes in the prior art more clearly, the drawings needed in the embodiments are briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application, and other drawings are available according to these drawings without creative work for ordinary people in the field.

FIG. 1 shows viral load of oropharyngeal swabs of B2 haplotype chicken, with n=7.

FIG. 2 illustrates antibody levels in serum, with n=4.

FIG. 3 illustrates changes of CD8α⁺ T cells in PBMC of B2 haplotype chickens after infection, with n=4.

FIG. 4 illustrates changes of CD4⁺ T cells in PBMC of B2 haplotype chickens after infection, with n=4.

FIG. 5 illustrates changes of proportions of CD4⁺ and CD8α⁺ T cells in PBMC of B2 haplotype chickens after infection; n=4.

FIG. 6 shows changes of CD⁴⁺/CD8α+ T cells in PBMC of B2 haplotype chickens after infection, n=4.

FIG. 7 represents expressions of natural immune related genes in PBMC of B2 haplotype chickens after H9N2 avian influenza virus (AIV) infection.

FIG. 8 represents expressions of cytotoxic T cells (CTLs)-related genes in PBMC of B2 haplotype chickens after H9N2 AIV infection.

FIG. 9 represents expressions of Th2-related genes in PBMC of B2 haplotype chickens after H9N2 AIV infection.

FIG. 10A, FIG. 10B and FIG. 10C show expression levels of interferon gamma (IFN-γ) of the splenocytes which are stimulated with peptide pool, while FIG. 10A shows ELISpot results of pool_1-pool_28, FIG. 10B shows ELISpot results of pool_29-pool_56, and FIG. 10C shows ELISpot results of pool_57-pool_85, with n=3 except in positive control.

FIG. 11 shows representative images of IFN-γ ELISpot responses of #1 chicken.

FIG. 12 shows representative images of IFN-γ ELISpot responses of #2 chicken.

FIG. 13 shows representative images of IFN-γ ELISpot responses of #3 chicken.

FIG. 14 shows secretion levels of IFN-γ in spleen lymphocytes of the #1 chicken, with three technical replicates of each peptide, except for the positive control.

FIG. 15 shows secretion levels of IFN-γ in spleen lymphocytes of the #2 chicken, with three technical replicates of each peptide, except for the positive control.

FIG. 16 shows secretion levels of IFN-γ in spleen lymphocytes of the #3 chicken, with three technical replicates of each peptide, except for the positive control.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Various exemplary embodiments of the present application are now described in detail and this detailed description should not be considered as limiting the present application, but should be understood as a more detailed description of certain aspects, features and embodiments of the present application.

It should be understood that the terms described in the present application are intended to describe particular embodiments only and are not intended to limit the present application. Furthermore, with respect to the range of values in the present application, it is to be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within a stated range is also included in the present application. The upper and lower limits of these smaller ranges may be independently included or excluded from the scope.

Unless otherwise stated, all technical and scientific terms used herein have the same meaning as is commonly understood by those of ordinary skill in the field described in the present application. Although the present application describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present application. All literature referred to in this specification is incorporated by reference for the purpose of disclosing and describing the methods and/or materials associated with said literature. In the event of conflict with any incorporated literature, the contents of this specification shall prevail.

Without departing from the scope or spirit of the present application, various improvements and variations can be made to specific embodiments of the specification of the present application, as will be apparent to those skilled in the art. Other embodiments derived from the specification of the present application are obvious to the skilled person. The specification and embodiments of the invention are only exemplary.

As used herein, the words “comprising”, “including”, “having”, “containing”, etc., are open-ended terms, i.e. meaning including but not limited to.

Terminology Explanation:

AIV: avian influenza virus; MHC I: class I major histocompatibility complex; ELISpot: enzyme-linked immune-absorbent spot; PBMC: peripheral blood mononuclear cells; SPF chicken: specific pathogens free chicken; CTL: cytotoxic T lymphocytes; IFN-γ: interferon-gamma; DPI: days post infection; EID₅₀: 50% embryo infective dose of chicken; FBS: fetal bovine serum; PMA+Ionomycin: phorbol myristoyl acetate and ionomycin.

Embodiment 1

1. Experimental Materials

1.1 Experimental Animals and Viruses

The chickens used in this experiment are 4-week-old B2 haplotype SPF chickens (BW/G3) purchased from the National Poultry Laboratory Animal Resource Center; the H9N2 subtype AIV (A/Chicken/Hunan/HN/2015) strain is isolated and stored in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control.

1.2 Main Experimental Reagents

Total RNA extraction kit is purchased from Jianshi Biotechnology Company; chicken peripheral blood lymphocyte separation liquid kit; chicken organ tissue mononuclear cell separation kit and red blood cell lysis buffer are purchased from Tianjin Haoyang Biological Manufacture Co., Ltd; ChamQ SYRB qPCR Master Mix are purchased from Nanjing Vazyme Biotech Co., Ltd.; Chicken IFN-γ ELISpot BASIC kit is purchased from Mabtech Company; flow antibodies including Anti-chicken CD3 antibody, Anti-chicken CD4 antibody, Anti-chicken CD8α antibody are purchased from Southern Biotech Company; PMA+Ionomycin and 3,3′,5,5′-tetramethylbenzidine (TMB) ELISpot chromogenic substrates are purchased from Dakewe Biotech Co., Ltd. of China; RPMI-1640 medium and Fetal Bovine Serum (FBS) Australian fetal bovine serum are purchased from GIBCO Company of the United States.

1.3 Preparation of Main Solution

(1) 1640 complete medium: 45 milliliters (mL) RPMI-1640 medium, 5 mL inactivated FBS and 500 microliters (μL) streptomycin (100×) are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.;

(2) flow buffer: 49 mL RPMI-1640 culture medium and 1 mL of inactivated FBS are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.;

(3) cells cryopreservation solution: 45 mL inactivated FBS and 5 mL dimethylsulfoxide (DMSO) are added into a 50 mL centrifuge tube, and mixed evenly at 4° C.

2 Experimental Methods

2.1 Virus Propagation

H9N2 AIV is melted and diluted 1,000 times in sterile PBS to obtain diluted virus solution, 9-11-day SPF chicken embryos are sterilized and placed on the biosafety cabinets, with each chick embryo allantoic cavity inoculated with 100 μL diluted virus solution, followed by sealing and continuing culture of the chick embryo for 24 hours (h); the survival of the chicken embryos is observed 24 h after the inoculation and the dead embryos are discarded; the remaining live embryos are continued to be cultured for 72 h to collect the virus, followed by aspirating the allantoic liquid into a centrifuge tube with a pipette, centrifuging the liquid at 4 degrees Celsius (° C.) for 10 minutes (min) at 2,000 revolutions per minute (rpm) to obtain a supernatant, passing the supernatant through a 0.22 micrometers (μm) filter membrane and sub-packing, storing at −80° C. for later use.

2.2 Determination of Hemagglutination Titer

The titer is determined with reference to Chinese national standards of the latest edition (GB/T 18936-2020).

2.3 Determination of EID₅₀

The 50% embryo infective dose (EID₅₀) of chicken embryo is determined as follows:

• • the virus solution after propagation is taken out and melted on ice, then diluted 10 times with PBS; the virus solution with a dilution of 10⁻⁴-10⁻⁹ is used to inoculate chicken embryos according to the method of 2.1, with 5 chicken embryos at each dilution and followed by culture 72 h; then, 25 μL allantoic liquid is collected from each chick embryo, the hemagglutination titer is determined by the method of 2.2, and the EID₅₀ is calculated by Spearman-Karber method.

2.4 Establishment of Animal Model of H9N2 Subtype AIV infection in B2 Haplotype Chickens

H9N2 AIV virus solution is diluted with sterile PBS to 10⁷ EID₅₀/200 μL; the experimental animals are divided into two groups, including B2 haplotype chicken experimental group and B2 haplotype chicken control group, with 7 chickens in each group. The experimental group is challenged intranasally and intratracheally, where 200 μL of virus is injected in the eyes of each animal with one drop in the left and one drop in the right eye firstly, then the rest of the virus is injected through the nasal cavity of one side; the control group is inoculated with equal volume of PBS in the same way. Swabs from the oropharyngeal and cloaca of chickens, peripheral anticoagulation and non-anticoagulation are collected and detected on 3, 5, 7, 9 and 11 DPI.

2.4.1 Detection of H9N2 AIV Shedding of Infected Chickens

The swabs collected on the sampling days are stored at −80° C. for unified detection. After the swab is taken out, it is melted on the ice, fully vortexed, and then the impurities are removed by centrifugation at 4° C. and 12,000 rpm for 5 min; then the supernatant is filtered through a 0.22 μm filter, and the EID₅₀ is determined according to 2.3, usually with a dilution of 10⁰-10⁻⁶, so as to evaluate the virus shedding.

2.4.2 Detection of Serum Antibody Levels

The collected non-anticoagulants are placed at room temperature until the serum is precipitated, and the serum is collected into a 1.5 mL centrifuge tube, followed by centrifugation at 4° C. and 2,000 rpm for 10 min to remove the red blood cells, and the remaining serum is used to detect the serum antibody level.

The antibody level is detected by hemagglutination inhibition (HI) test, with reference to the latest edition of national standard (GB/T 18936-2020).

2.4.3 Detection of T Cell Percentage in Chicken PBMC

PBMC is separated according to the instruction of the kit, and appropriate cells are taken for flow staining; the following descriptions are based on 10⁶ cells per tube: cells are added in flow tubes, then 1 mL of flow Buffer is added, followed by centrifugation at 440 g for 6 min, and CD3, CD4 and CD8 antibodies are diluted in the dark according to the recommended concentrations in the instruction; the supernatant is discarded after centrifugation, with 100 μL of diluted antibody added to each tube for re-suspension, and incubated for 30 min at 4° C. in the dark; then, 1 mL Buffer is added, and centrifuged at 440 g for 6 min, with precipitate resuspended with 250 μL flow Buffer; the data are collected by an up-flow cytometry and analyzed by a FlowJo software.

2.4.4 Expressions of Immune-Related Genes in Chicken PBMC by Fluorescence Quantitative Polymerase Chain Reaction (PCR) Detection

The total RNA is extracted according to the total RNA extraction kit from Jianshi Biotechnology Company, briefly, taking an appropriate amount of cells, centrifuging at 440 g for 6 min, discarding the supernatant, adding 1 mL of TRIzol then whirling; then adding equal volume of anhydrous ethanol, mixing well, transferring the liquid to a No.2 column, centrifuging for 1 min, and removing the filtrate; adding 400 μL RNA washing solution 2 to the column, centrifuging for 1 min, and discarding the filtrate; add 80 μL of DNase I reaction solution to the column, reacting at room temperature for 15 min, then adding 400 μL of RNA washing solution 1, centrifuging for 1 min, and discarding the filtrate; placing for 2 min, transferring the No.2 column to a centrifugal tube without RNase, adding 50 μL RNase-free water preheated to 70° C. in advance, standing for 2 min, centrifuging for 1 min to elute RNA, and detecting the concentration with ultra-micro spectrophotometer, and storing at −80° C.

RNA is reverse-transcribed according to the system in Table 1 following a reverse transcription procedure as follows: performing reverse transcription PCR at 37° C. for 15 min, inactivating at 85° C. for 5 s, and storing at 4° C.

TABLE 1
Reverse Transcription System

	Components	Dosage
	5 × M-MLV-Mix	4 μL
	RNA	10 μL (less than 1,000 ng)
	Sterilized water	6 μL
	Total volume	20 μL

The reverse-transcribed complementary deoxyribonucleic acid (cDNA) is amplified by fluorescence quantitative PCR to detect the changes of immune-related genes in PBMC; the target genes and primers detected are as shown in Table 2, and the system is shown in Table 3. Reaction procedure: pre-denaturation at 95° C. for 30 s; cyclic reaction at 95° C., 10 s, 60° C. and 30 s, a total of 40 cycles; dissolution curve analysis 95° C., 15 s, 60° C., 60 s, 95° C., 15 s. The results are analyzed by 2^−ΔΔCt method with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene as internal reference.

TABLE 2
qPCR25 Primers for Immune Related Genes

Gene name	Primer sequence (5′-3′)	Sequence ID

GAPDH	F-Primer	GAACATCATCCCAGCGTCCA	SEQ ID NO. 5
	R-Primer	CGGCAGGTCAGGTCAACAAC	SEQ ID NO. 6
Granzyme	F-Primer	CGGGAAGCAACTGTTGAAAT	SEQ ID NO. 7
K	R-Primer	GAGTCTCCCTTGCAAGCATC	SEQ ID NO. 8
Perforin	F-Primer	ATGGCGCAGGTGACAGTGA	SEQ ID NO. 9
	R-Primer	TGGCCTGCACCGGTAATTC	SEQ ID NO. 10
IFN-γ	F-Primer	CCTCCAACACCTCTTCAACATG	SEQ ID NO. 11
	R-Primer	TGGCGTGCGGTCAAT	SEQ ID NO. 12
TNF-α	F-Primer	GCTGTTCTATGACCGCCCAGTT	SEQ ID NO. 13
	R-Primer	AACAACCAGCTATGCACCCCA	SEQ ID NO. 14
IL-1β	F-Primer	GGTCAACATCGCCACCTACA	SEQ ID NO. 15
	R-Primer	CATACGAGATGGAAACCAGCAA	SEQ ID NO. 16
IL-2	F-Primer	GCTAATGACTACAGCTTATGGAGCA	SEQ ID NO. 17
	R-Primer	TGGGTCTCAGTTGGTGTGTAGAG	SEQ ID NO. 18
NK lysin	F-Primer	GATGGTTCAGCTGCGTGGGATGC	SEQ ID NO. 19
	R-Primer	CTGCCGGAGCTTCTTCAACA	SEQ ID NO. 20
HMG-2	F-Primer	AGAGCACAAGAAGAAGCAC	SEQ ID NO. 21
	R-Primer	GTCTTTTAGGAGCGTTGGGGTC	SEQ ID NO. 22
MHC-I	F-Primer	AAGAAGGGGAAGGGCTACAA	SEQ ID NO. 23
	R-Primer	AAGCAGTGCAGGCAAAGAAT	SEQ ID NO. 24
IFN-α	F-Primer	GACAGCCAACGCCAAAGC	SEQ ID NO. 25
	R-Primer	GTCGCTGCTGTCCAAGCATT	SEQ ID NO. 26
IFN-β	F-Primer	GCCCACACACTCCAAAACACTG	SEQ ID NO. 27
	R-Primer	TTGATGCTGAGGTGAGCGTTG	SEQ ID NO. 28
IL-6	F-Primer	AAATCCCTCCTCGCCAATCT	SEQ ID NO. 29
	R-Primer	CCCTCACGGTCTTCTCCATAAA	SEQ ID NO. 30
IL-10	F-Primer	AGCAGATCAAGGAGACGTTC	SEQ ID NO. 31
	R-Primer	ATCAGCAGGTACTCCTCGAT	SEQ ID NO. 32
TLR3	F-Primer	ACAATGGCAGATTGTAGTCACCT	SEQ ID NO. 33
	R-Primer	GCACAATCCTGGTTTCAGTTTAG	SEQ ID NO. 34
TLR7	F-Primer	TCTGGACTTCTCTAACAACA	SEQ ID NO. 35
	R-Primer	AATCTCATTCTCATTCATCATCA	SEQ ID NO. 36
MHC-I	F-Primer	AAGAAGGGGAAGGGCTACAA	SEQ ID NO. 37
	R-Primer	AAGCAGTGCAGGCAAAGAAT	SEQ ID NO. 38
MHC-II	F-Primer	CTCGAGGTCATGATCAGCAA	SEQ ID NO. 39
	R-Primer	TGTAAACGTCTCCCCTTTGG	SEQ ID NO. 40
IL-4	F-Primer	TCGAGGAGTGACGGGTG	SEQ ID NO. 41
	R-Primer	ACTATCCGGATGCTCTCCATC	SEQ ID NO. 42
IL-5	F-Primer	GGAACGGCACTGTTGAAAAATAA	SEQ ID NO. 43
	R-Primer	TTCTCCCTCTCCTGTCAGTTGTG	SEQ ID NO. 44
IL-13	F-Primer	CTGCCCTTGCTCTCCTCTGT	SEQ ID NO. 45
	R-Primer	CCTGCACTCCTCTGTTGAGCTT	SEQ ID NO. 46
Granzyme	F-Primer	ACTCATGTCGAGGGGATTCA	SEQ ID NO. 47
A	R-Primer	TGTAGACACCAGGACCACCA	SEQ ID NO. 48
MDA5	F-Primer	GGACGACCACGATCTCTGTGT	SEQ ID NO. 49
	R-Primer	CACCTGTCTGGTCTGCATGTTATC	SEQ ID NO. 50
CXCLi1	F-Primer	AACTCCGATGCCAGTG	SEQ ID NO. 51
	R-Primer	TTGGTGTCTGCCTTGT	SEQ ID NO. 52
CXCLi2	F-Primer	CATCATGAAGCATTCCATCT	SEQ ID NO. 53
	R-Primer	CTTCCAAGGGATCTTCATTT	SEQ ID NO. 54
TGF-β3	F-Primer	TCTTTACATTGACTTCCGAC	SEQ ID NO. 55
	R-Primer	TCCTCCCAACATAGTACAAG	SEQ ID NO. 56
MX1	F-Primer	AAGCCTGAGCATGAGCAGAA	SEQ ID NO. 57
	R-Primer	TCTCAGGCTGTCAACAAGATCAA	SEQ ID NO. 58
OASL	F-Primer	AGATGTTGAAGCCGAAGTACCC	SEQ ID NO. 59
	R-Primer	CTGAAGTCCTCCCTGCCTGT	SEQ ID NO. 60
ISG12-2	F-Primer	TCAATGGGTGGCAAAGGAG	SEQ ID NO. 61
	R-Primer	TACAGGGAGAGCAAAGAAGAGAAGA	SEQ ID NO. 62
IFIT5	F-Primer	CAGAATTTAATGCCGGCTATGC	SEQ ID NO. 63
	R-Primer	TGCAAGTAAAGCCAAAAGATAAGTGT	SEQ ID NO. 64
USP18	F-Primer	CAACGTGGGAAGAGGAGAAA	SEQ ID NO. 65
	R-Primer	ACTTCATGAGCGGAGAAGGA	SEQ ID NO. 66
IRF3/7	F-Primer	ACTGACCAGCCCAGGAACTCT	SEQ ID NO. 67
	R-Primer	AAGGCTTTCCCAACCACAAA	SEQ ID NO. 68
SST	F-Primer	GGTCCACGGTTATGGTGAAAG	SEQ ID NO. 69
	R-Primer	GGTCAGAAATCACAACTCAAGCA	SEQ ID NO. 70
KHSRP	F-Primer	CAGCGGGGAAATGATTAAGAAG	SEQ ID NO. 71
	R-Primer	TTTGTGTGTGGGGATGGAGA	SEQ ID NO. 72
PARP	F-Primer	ATTGTGGAGGAGCTGGGAGGAA	SEQ ID NO. 73
	R-Primer	AGGCTTGCTGCACTTCCCATC	SEQ ID NO. 74

TABLE 3
Fluorescence Quantitative System

	Reagent	Volume

	2 × ChamQ Universal	10	μL
	SYBR qPCR Mix
	F-Primer	0.4	μL
	R-Primer	0.4	μL
	cDNA	2	μL
	ddH2O	7.2	μL

2.5 Screening Polypeptide Epitopes with Immunogenicity

According to the binding motif (X-A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) of MHC class I molecular in B2 haplotype chickens determined in the National and Regional Joint Engineering Laboratory for Medicament of Zoonosis Prevention and Control, candidate polypeptide epitopes against H9N2 subtype AIV are screened, then the peptides are synthesized by Shanghai Top-Peptide Biotechnology Co., Ltd., with a purity of 95%, and each peptide is synthesized for 5 mg.

2.6 Immunogenicity Detection of Candidate Polypeptides

2.6.1 ELISpot Experiment for Detecting Immunogenicity of Peptide Pool

The synthesized polypeptide is dissolved in 200 μL DMSO, and then stored at −80° C.; five peptides are mixed into a pool, named from pool_1 to pool_85 respectively, and the immunogenicity is detected by ELISpot experiment, with experimental operation referring to the instructions of Chicken IFN-γ ELISpot BASIC Kit, the details are as follows:

the first day:

• • (1) the PVDF membrane in the cell wells is activated by 15 μL of 35% ethanol per well using a multichannel pipette for no more than 1 min; then each well is added with 200 μL sterile water for washing, and repeated for 4 times; the monoclonal antibody against chicken IFN-γ is diluted with sterile water at 1:33, and 100 μL is added to each well, and coated at 4° C. overnight;

the second day:

• • (2) the coating solution is discarded, and each well is washed with 200 μL sterile PBS for 4 times;
  • (3) each well is added with 200 μL 1640 complete culture medium and incubated at room temperature for 1-2 h;
  • (3) the solution is discarded, 100 μL spleen lymphocyte suspension (containing 3.5×10⁵ cells) is added to each well: at the same time, peptide library (final concentration of 10 micrograms per microliter (m/mL) for each peptide) is added to the experimental group, an equal volume of DMSO is added to the negative control group, and 10 μL of PMA+Ionomycin mixture from the Dakewe Biotech Co., Ltd. is added into the positive control group;
  • (4) after all the samples are added, the cell plate is placed into a 37° C. cell incubator containing 5% CO₂ for culture of at least 18 h;

the third day:

• • (5) after the culture, the culture medium and cells are discarded, and each well is washed five times by 200 μL sterile PBS; each well is added with 100 μL PBS containing 0.5% FBS and 1 μg/mL biotin-labeled detection antibody, and incubated for 2 h at room temperature; then the liquid is shaken off and 200 μL sterile PBS is added to each well for washing for 5 times;
  • (6) each well is added with 100 μL diluted streptavidin-labeled horseradish peroxidase (HRP) and incubated at room temperature for 1 h; then the liquid is discarded and each well is added with 200 μL sterile PBS for washing for 5 times;
  • (7) each well is added with 100 μL TMB chromogenic solution, and the reaction is stopped by washing the cell plate with ultrapure water until obvious spots appear at the bottom, then the cell plate is dried and counted in an automatic plate reader, and the number of spots is statistically analyzed.

2.6.2 ELISpot Experiment for Detecting Immunogenicity of Individual Peptide

The peptide pools in 2.6.1 that can significantly stimulate cells to produce spots are selected and detected for individual peptide by ELISpot experiment, the methods are the same as in 2.6.1.

2.7 Data Analysis

All the experimental data are statistically analyzed by using the software GraphPad Prism 8, in which ns means P>0.05, where the difference is not significant. * means P<0.05, with significant difference; ** means P<0.01, where the difference is extremely significant; *** means P<0.001, indicating an extremely significant difference, and **** means P<0.0001, where the difference is extremely significant.

3 Results

3.1 Detection of Detoxification of Infected Chicken

After H9N2 AIV infection of B2 haplotype chickens, swabs are collected and the virus shedding is detected according to 2.4.1. As shown in FIG. 1, in the H9N2 AIV-infected group, the viral load of oropharyngeal swabs peaked at 3 DPI, had fallen off since SDPI (p<0.0/), and disappeared at 11 DPI. The cloacal swabs were found to be positive at 3 DPI and SDPI and negative at 7 DPI in table 4, while control groups are all tested negative (the data is not included).

TABLE 4
The virus shedding of cloacal swabs in B2 Haplotype Chickens

	Groups	3DPI	5DPI	7DPI
	B2 chicken	3.5, 4.3 (2/7)	1.5, 1.5 (2/7)	(0/7)
	Note:
	the figures outside brackets indicate the virus shedding of cloacal swabs of infected chickens, which is the value of log10EID50. The figures inside brackets/before brackets indicate the number of chickens that have been detoxified, and the figures after/indicate the total number of chickens.

3.2 Detection of Serum Antibody Level in Infected Chickens

According to the results as shown in FIG. 2, the antibody levels are negative at 3 DPI (all less than 2 wells), all chickens tested at 5 DPI turn positive, and the HI antibody levels continue to rise until 11 DPI, indicating that the humoral immunity is initiated on 5 DPI, and the decrease the virus shedding in cloacal swabs from 5 DPI is associated with an increase in antibody levels. The control groups are all tested negative for antibody (the data is not included).

3.3 Changes of T Cell Subtypes in Chicken PBMC after Infection

In order to detect the immune response of T cells in B2 haplotype chickens after challenge, peripheral blood of chickens is collected from jugular vein according to the experimental arrangement, and PBMC is isolated and stained with flow antibody. As can be seen from FIG. 3, the proportion of CD8⁺ T cells significantly increased in the H9N2 AIV-infected group compared to that in the control group at 5 DPI, 7 DPI and 9 DPI (P<0.001); the results show that significant CD8+ T cell proliferation is detectable in chicken PBMC from day 5 after H9N2 AIV infection of B2 haplotype chickens and persists until 9 DPI, suggesting that virus clearance from 5 DPI is associated not only with elevated antibody levels but also subjects to an important effect of CD8+ T cell immune response.

The changes of CD4⁺ T cell subtypes are illustrated in FIG. 4. After H9N2 AIV infection of B2 haplotype chickens, the proportion of CD4⁺ T cells decreases significantly (P<0.05) at 5 DPI, 7 DPI, 9 DPI and returns to a normal level at 11 DPI; the proportion changes of CD4⁺CD8α⁺ double positive T cells in PBMC from B2 haplotype chickens after challenge are shown in FIG. 5, with no statistical difference in the proportion of CD4⁺CD8α⁺ double positive T cells in the challenge group compared to that in the control group. Moreover, the changes of CD4⁺/CD8α⁺ T cells in B2 haplotype chicken PBMC after the challenge are shown in FIG. 6, and the CD4⁺/CD8α⁺ T cell ratios in the 5 DPI, 7 DPI and 9 DPI challenge groups are significantly lower than those in the control group, indicating that the organism is in an immunosuppressed state at this stage.

3.4 Expression of Immune-Related Genes in PBMC of B2 Haplotype Chickens after Infection

In order to further verify the influence of immune response in the process of H9N2 AIV infecting B2 haplotype chickens, the changes of mRNA expression of important immune genes in PBMC after 5 days of in vivo infection are detected by fluorescence quantitative PCR, which mainly includes three parts: natural immune-related genes, CTLs genes and Th2 genes.

In the innate immunity gene fraction (FIG. 7), the expressions of the antiviral genes interferon-stimulated gene 12-2 (ISG12-2), 2′,5′ -oligoadenylate synthetase-like (OASL), interferon-induced proteins with tetratricopeptide repeats 5 (IFIT5), (ubiquitin specific peptidase 18) USP18 and myxovirus resistance 1 (MX1) significantly increased in the 5 DPI infected group compared to those in the control groups (P<0.05). As can be seen from the CTLs gene fraction (FIG. 8), the expressions of Granzyme k, IFN-γ, NK lysin, Poly-(ADP-ribose) Polymerase (PARP) increased significantly (P<0.05); and there no obvious changes of expression as detected in the Th2 gene (FIG. 9). Taken together with the increased proportion of CD8α⁺ T cells as shown in 3.3, it is further suggested that AIV infection has successfully activated the cytotoxic T cell immune response in B2 haplotype chickens. In summary, these results suggest that the model of H9N2 subtype AIV infection inducing cellular immune response in B2 haplotype SPF chickens (BW/G3) is successfully established.

3.5 Screening of Candidate Polypeptide Epitopes with Immunogenicity

According to the binding motif (A/V/I/L/P/S/G-X-X-X-X-X-X-V/I/L) of MHC class I molecular of B2 haplotype chicken, the candidate polypeptide epitopes for H9N2 subtype AIV are screened, and the screened peptides are shown in Table 5.

TABLE 5
Candidate Polypeptides with Immunogenicity
Screened According to the Motif

Pool				Pool
serial	Peptide	Peptide	Sequence	serial	Peptide	Peptide	Sequence
number	name	sequence	ID	number	name	sequence	ID

Pool_1	P1	SAKEAQDVI	75	Pool_2	P6	LIGQGDVVL	216
	P2	SAVLRGFLI	76		P7	ELVRKTRFL	217
	P3	PIDNVMGMI	77		P8	VLTGNLQTL	218
	P4	SLIIAARNI	78		P9	ILRKATKRL	219
	P5	VLVNTYQWI	79		P10	AVKGIGTMV	1
Pool_3	P11	DVSFQGRGV	2	Pool_4	P16	MVGRRATAI	80
	P12	GVRVSKMGV	355		P17	KVLFQNWGI	81
	P13	RVRDQRGNV	356		P18	LVNTYQWII	82
	P14	NVLLSPEEV	357		P19	NVRGSGMRI	83
	P15	DVLGTFDTV	358		P20	YPITADKRI	84
Pool_5	P21	RLNPMHQLL	220	Pool_6	P26	IVVRGNSPV	359
	P22	VLGKDAGAL	221		P27	NVLIGQGDV	360
	P23	SVYIEVLHL	222		P28	GPVHFRNQV	361
	P24	SVKREEEVL	223		P29	DPDEGTAGV	362
	P25	FVNRANQRL	224		P30	TSTVHYPKV	363
Pool_7	P31	FPNEVGARI	85	Pool_8	P36	NVMGMIGIL	225
	P32	MSQSRTREI	86		P37	VVVSIDRFL	226
	P33	VSGKDEQSI	87		P38	AVLRGFLIL	227
	P34	YSSSMMWEI	88		P39	APLMVAYML	228
	P35	DSQTATKRI	89		P40	GPALSINEL	229
Pool_9	P41	LSAKEAQDV	364	Pool_10	P46	AGGTSSVYI	90
	P42	SSVKREEEV	365		P47	IGGVRMVDI	91
	P43	QSIAEAIIV	366		P48	GTFDTVQI	92
	P44	YSSTERVVV	367		P49	KGEKANVLI	93
	P45	VSIDRFLRV	368		P50	DAGSDRVMV	94
Pool_11	P51	TSSVYIEVL	230	Pool_12	P56	GGEVRNDDV	369
	P52	VSADPLASL	231		P57	EGYEEFTMV	370
	P53	QSRMQFSSL	232		P58	WGIEPIDNV	371
	P54	ESAVLRGFL	233		P59	RGSGMRIVV	372
	P55	LSINELSNL	234		P60	IGQGDVVLV	373
Pool_13	P61	VGRRATAIL	95	Pool_14	P66	GGVRMVDIL	235
	P62	KATKRLIQL	96		P67	NLQTLKLRV	236
	P63	KATKRLTVL	97		P68	VLFQQMRDV	237
	P64	TAGVESAVL	98		P69	VLIGQGDVV	238
	P65	DINPGHADL	99		P70	RVMVSPLAV	239
Pool_15	P71	VAYMLEREL	374	Pool_16	P76	RILTSESQL	100
	P72	TGNLQTLKL	375		P77	DICKAAMGL	101
	P73	LAKGEKANV	376		P78	MIKAVRGDL	102
	P74	RATVSADPL	377		P79	EINGPESVL	103
	P75	SADPLASLL	378		P80	LIIAARNIV	104
Pool_17	P81	PVAGGTSSV	240	Pool_18	P86	LLRTAVGQV	379
	P82	AVRGDLNFV	241		P87	ASICTHLEV	380
	P83	EAQDVIMEV	242		P88	NSICNTTGV	381
	P84	CLLQSLQQI	243		P89	VSRPMFLYV	382
	P85	SLQQIESMI	244		P90	ESRKLLLIV	383
Pool_19	P91	MAWTVVNSI	105	Pool_20	P96	DLEGLYEAI	245
	P92	AAMDDFQLI	106		P97	GVTRREVHI	246
	P93	KIKSEKTHI	107		P98	QVLSELQDI	247
	P94	RIKTRLFTI	108		P99	DPSHEGEGI	248
	P95	IIKPHKKGI	109		P100	EPRSLSCWI	249
Pool_21	P101	MATKADYTL	384	Pool_22	P106	EIGEDVAPI	110
	P102	CAAMDDFQL	385		P107	ALLKHRFEI	111
	P103	LAKSVFNSL	386		P108	LLKHRFEII	112
	P104	SAESRKLLL	387		P109	EIGVTRREV	113
	P105	NASWFNSFL	388		P110	PIGESPKGV	114
Pool_23	P111	KSEKTHIHI	250	Pool_24	P116	EITGTVRRL	389
	P112	QSERGEETI	251		P117	FIIKGRSHL	390
	P113	MSKEVNARI	252		P118	SIGKVCRTL	391
	P114	SSWVELDEI	253		P119	LINDPWVLL	392
	P115	DGFEPNGCI	254		P120	SLPPNFSSL	393
Pool_25	P121	SLENFRAYV	115	Pool_26	P126	EGIPLYDAI	255
	P122	KLSQMSKEV	116		P127	FGWKEPNII	256
	P123	ELDEIGEDV	117		P128	FLLMDALKL	257
	P124	HLRNDTDVV	118		P129	PGTFDLEGL	258
	P125	WGMEMRRCL	119		P130	ESSVKEKDL	259
Pool_27	P131	FLKTTPRPL	394	Pool_28	P136	IGKVCRTLL	120
	P132	NSLYASPQL	395		P137	CVLEIGDML	121
	P133	FSAESRKLL	396		P138	IVQALRDNL	122
	P134	TGVEKPKFL	397		P139	ESGDPNALL	123
	P135	KGINPNYLL	398		P140	CSORSKFLL	124
Pool_29	P141	VLEIGDMLL	260	Pool_30	P146	KGVYINTAL	399
	P142	KLLLIVQAL	261		P147	YLLTWKQVL	400
	P143	NLEPGTFDL	262		P148	SITIERMVL	401
	P144	CLINDPWVL	263		P149	VGIDPFRLL	402
	P145	GVYINTALL	264		P150	GIGTMVMEL	403
Pool_31	P151	TAGLTHLMI	125	Pool_32	P156	LSDNEGRLI	265
	P152	AAGAAVKGI	126		P157	TSDMRTEII	266
	P153	VAYERMCNI	127		P158	EGRLIONSI	267
	P154	LILYDKEEI	128		P159	DGKWVRELI	268
	P155	LIRMIKRGI	129		P160	IGTMVMELI	269
Pool_33	P161	LIFLARSAL	404	Pool_34	P166	RLIQNSITI	130
	P162	KLSDNEGRL	405		P167	QLSTRGVQI	131
	P163	HLMIWHSNL	406		P168	ELRSRYWAI	132
	P164	FLARSALIL	407		P169	NLPFERATI	133
	P165	CLPACVYGL	408		P170	SVGRMVSGI	134
Pool_35	P171	NATEIRASV	270	Pool_36	P176	LVGIDPFRL	409
	P172	SALILRGSV	271		P177	CSLMQGSTL	410
	P173	PACVYGLAV	272		P178	GSVAHKSCL	411
	P174	SAAFEDLRV	273		P179	NGEDATAGL	412
	P175	DPKKTGGPI	274		P180	PGNAEIEDL	413
Pool_37	P181	MVMELIRMI	135	Pool_38	P186	VSGIGRFYI	275
	P182	RSGAAGAAV	136		P187	NPAHKSQLV	276
	P183	ISVQPTFSV	137		P188	NSITIERMV	277
	P184	RGQLSTRGV	138		P189	RASAGQISV	278
	P185	DATAGLTHL	139		P190	IAIGSVSLI	279
Pool_39	P191	KADTRVLFI	414	Pool 40	P196	ASGKADTRV	140
	P192	AIGSVSLII	415		P197	SSYVCSGLV	141
	P193	AIICLLMQI	416		P198	RSGYETFRV	142
	P194	SIGSWSKNI	417		P199	SGYETFRVV	143
	P195	YINMADYSI	418		P200	SGYSGIFSV	144
Pool_41	P201	RVWWTSNSI	280	Pool_42	P206	HLGTKQVCI	419
	P202	NPNQKIIAI	281		P207	VLFIREGKI	420
	P203	APFSKDNSI	282		P208	SVSLIIAII	421
	P204	GSVSLIIAI	283		P209	QVMPCEPII	422
	P205	NSTIIEREI	284		P210	TVHLNSTII	423
Pool_43	P211	RGRPQEPRV	145	Pool_44	P216	GSNRPILYI	285
	P212	FALGQGTTL	146		P217	KSQVNRQVI	286
	P213	IIAIGSVSL	147		P218	FSVEGKKCI	287
	P214	LIIAIICLL	148		P219	GSWPDGANI	288
	P215	AILTTTMTL	149		P220	WAFDDGNDV	289
Pool_45	P221	ILERNTVHL	424	Pool_46	P226	SLIIAIICL	150
	P222	IAIGSVSLI	425		P227	MATTTNPLI	151
	P223	ASIIYDGML	426		P228	DLESLMEWI	152
	P224	ESSYVCSGL	427		P229	ILSPLAKGI	153
	P225	IGSWSKNIL	428		P230	ALASCMGLI	154
Pool_47	P231	GANINFMPV	290	Pool_48	P236	SGKADTRVL	429
	P232	FSKDNSIRL	291		P237	PLAGSAQHV	430
	P233	LVCATCEQI	292		P238	CSNPTNNQV	431
	P234	PSGPLKAEI	293		P239	LSAGGDIWV	432
	P235	LAKGILGFV	294		P240	PIILERNTV	433
Pool_49	P241	EVETYVLSI	155	Pool_50	P246	TAEGALGLV	295
	P242	DPNNMDKAV	156		P247	VASQARQMV	296
	P243	LGFVFTLTV	157		P248	LSIIPSGPL	297
	P244	VIAANIIGI	158		P249	RGLORRRFV	298
	P245	NIIGILHLI	159		P250	KAVKLYKKL	299
Pool_51	P251	CINGTCAVV	434	Pool_52	P256	GILHLILWI	160
	P252	TLLMNELGV	435		P257	WIIRNWETV	4
	P253	CLLMQIAIL	436		P258	PLVIAANII	161
	P254	QAYQNRMGV	437		P259	DPLVIAANI	162
	P255	EIAQRLEDV	438		P260	SGSSDPLVI	163
Pool_53	P261	GALASCMGL	300	Pool_54	P266	LIYNRMGTV	439
	P262	GILGFVFTL	301		P267	LIRHENRMV	440
	P263	ALSYSTGAL	302		P268	LLTEVETYV	441
	P264	LSPLAKGIL	303		P269	SSTGLKDDL	442
	P265	CSGSSDPLV	304		P270	LILWILDRL	443
Pool_55	P271	RIQIFPDTI	164	Pool_56	P276	IAANIIGIL	305
	P272	RIKSNGNLI	165		P277	IIGILHLIL	306
	P273	LIAPWYGHI	166		P278	YIVERPSAV	307
	P274	KITSKVNNI	167		P279	GIKSLKLAV	308
	P275	KIDDQIQDI	168		P280	AIDKITSKV	309
Pool_57	P281	ILHLIL WIL	444	Pool_58	P286	PLILDTCTI	169
	P282	DGHFVNIEL	445		P287	RLNMINNKI	170
	P283	TASLITILL	446		P288	LVNGLMGRI	171
	P284	CATSLGHPL	447		P289	EVETRLNMI	172
	P285	IAPWYGHIL	448		P290	VPSRSSRGI	173
Pool_59	P291	JIDHEFSEV	310	Pool_60	P296	IAMGFAAFL	449
	P292	KILTIYSTV	311		P297	EINRTFKPL	450
	P293	SLITILLVV	312		P298	RINYYWSVL	451
	P294	TLTENNVPV	313		P299	YIGIKSLKL	452
	P295	PLIGPRPLV	314		P300	FIEGGWSGL	453
Pool_61	P301	VSNADKICI	174	Pool_62	P306	DLKRGSCTV	315
	P302	SSARSYQRI	175		P307	KLAVGLRNV	316
	P303	LSGESHGRI	176		P308	TLDEHDANV	317
	P304	SSRGIFGAI	177		P309	NVNNLYNKV	318
	P305	ESKLERQKI	178		P310	ASLITILLV	319
Pool_63	P311	DIWAYNAEL	454	Pool_64	P316	KPLIGPRPL	179
	P312	VLLENQKTL	455		P317	GPRPLVNGL	180
	P313	PVTHAKELL	456		P318	NSTETVDTL	181
	P314	SVLKPGQTL	457		P319	TSLGHPLIL	182
	P315	VPVTHAKEL	458		P320	KSLKLAVGL	183
Pool_65	P321	QSTNSTETV	320	Pool_66	P326	GGREWSYIV	459
	P322	YSTVASSLV	321		P327	NGLCYPGNV	460
	P323	AATALANTI	322		P328	VPAEMLANI	461
	P324	TANESGRLI	323		P329	LPSFGVSGI	462
	P325	EAMVSRARI	324		P330	NPFVSHKEI	463
Pool_67	P331	ESEGTYKIL	184	Pool_68	P336	AIATPGMQI	325
	P332	NGMLCATSL	185		P337	WIPKRNRSI	326
	P333	YGNPSCDLL	186		P338	RIDFESGRI	327
	P334	SGESHGRIL	187		P339	EIMKICSTI	328
	P335	HGRILKTDL	188		P340	FLEESHPGI	329
Pool_69	P341	NSCLETMEI	464	Pool_70	P346	EGTYKILTI	189
	P342	LSTVLGVSI	465		P347	WAYNAELLV	190
	P343	QSSDDFALI	466		P348	GSCRCNICI	191
	P344	SSYRRPVGI	467		P349	LGGREWSYI	192
	P345	TGAPQLNPI	468		P350	TLLFLKVPV	193
Pool_71	P351	RLNKRSYLI	330	Pool_72	P356	RGKLKRRAI	469
	P352	ILNTSQRGI	331		P357	DGGPNLYNI	470
	P353	FVEALARSI	332		P358	TALANTIEV	471
	P354	AVATTHSWI	333		P359	LIDFLKDVV	472
	P355	NPRMFLAMI	334		P360	QIRGFVYFV	473
Pool_73	P361	RLIDFLKDV	194	Pool_74	P366	EIDSVNNAV	335
	P362	KLEQSGLPV	195		P367	SSDDFALIV	336
	P363	NPTLLFLKV	196		P368	ESADMSIGV	337
	P364	RPVGISSMV	197		P369	VSHKEIDSV	338
	P365	KSMKLRTQV	198		P370	PSSSYRRPV	339
Pool_75	P371	TIGKKKQRL	474	Pool_76	P376	QLNPIDGPL	199
	P372	ISSMVEAMV	475		P377	LLIDGTASL	200
	P373	MSRDWLMLI	3		P378	VLGVSILNL	201
	P374	SGYAQTDCV	476		P379	NLHIPEVCL	202
	P375	PGMQIRGFV	477		P380	DVNPTLLFL	203
Pool_77	P381	TATREGKHI	340	Pool_78	P386	LARSICEKL	478
	P382	RAFTDEGAI	341		P387	VIFNRLEAL	479
	P383	TLKANFSVI	342		P388	AIVGEISPL	480
	P384	RSSTLGLDI	343		P389	RLRRDQKSL	481
	P385	SIRLMDRCL	344		P390	SLRGRSSTL	482
Pool_79	P391	CVLEAMAFL	204	Pool_80	P396	CIRMDQAIV	345
	P392	FVANFSMEL	205		P397	QVDCFLWHV	346
	P393	LVSDGGPNL	206		P398	LPGHTDKDV	347
	P394	QPEWFRNVL	207		P399	AIVDKNITL	348
	P395	GPATAQMAL	208		P400	EGKHIVERI	349
Pool_81	P401	DVKNAIEVL	483	Pool_82	P406	IPKRNRSIL	209
	P402	SSFQVDCFL	484		P407	ESGRLIDFL	210
	P403	ASVPAPRYL	485		P408	KICSTIEEL	211
	P404	RGRSSTLGL	486		P409	KIEKIRPLL	212
	P405	QLSOKFEEI	487		P410	QLRSSSEDL	213
Pool_83	P411	SSEDLNGMI	350	Pool_84	P416	LLLEVEQEI	488
	P412	QALQLLLEV	351		P417	TVSSFQDIL	489
	P413	EIR WLIEEV	352		P418	EAAMRMGDL	490
	P414	KLFSKQEWI	353		P419	LIEEVRHRL	491
	P415	RVGMHKRIV	354		P420	EIRTFSFQL	492
Pool_85	P421	SLKLYRDSL	214
	P422	RIVYWKQWL	215

3.6 Immunogenicity Detection of Candidate Peptides

3.6.1 ELISpot assay for Immunogenicity Detection of Peptide Pools

The synthesized peptides are mixed according to the requirement of five peptides as a pool, and the spleen lymphocytes of B2 haplotype chickens infected with H9N2 AIV for 28 days are stimulated respectively. As shown in FIGS. 10A-10C, it is found by statistical analysis that pool_2, pool_3, pool_52 and pool_75 significantly stimulate the production of IFN-γ spots by splenic lymphocytes, indicating the presence of immunogenic epitopes in the peptides that constitutes the above-mentioned peptide pools.

3.6.2 ELISpot Assay for Immunogenicity Detection of Polypeptide

FIGS. 11-13 suggest that a single peptide that is immunogenic may also stimulate IFN-γ production by splenic lymphocytes; according to the criteria provided in the reference (Identification of novel avian influenza virus derived CD8⁺ T-cell epitopes), a peptide that causes significant IFN-γ production in at least 2 of 3 chickens is considered immunogenic compared to a negative control, and four peptides, P10, P11, P373 and P257, satisfy this criteria and may be considered as B2 haplotype restrictive H9N2 AIV T-cell epitopes, as shown in FIGS. 14-16 (Shown in Table 6).

TABLE 6
Epitope Information for Four H9N2 Subtype AIV
T Cells Targeting Haplotype B2 Chickens

		Protein in
		which the
Polypeptide	Polypeptide	polypeptide	Protein
name	sequence	is located	sites

P10	AVKGIGTMV	NP	182-190
	(SEQ ID NO. 1)
P11	DVSFQGRGV	NP	455-463
	(SEQ ID NO. 2)
P373	MSRDWLMLI	NS1	98-106
	(SEQ ID NO. 3)
P257	WIIRNWETV	PB2	552-560
	(SEQ ID NO. 4)

The above-mentioned embodiments only describe the preferred mode of the present application, and do not limit the scope of the present application. Under the premise of not departing from the design spirit of the present application, various modifications and improvements made by ordinary technicians in the field to the technical scheme of the present application shall fall within the protection scope determined by the claims of the present application.